CN107966436A - A kind of visible sensor of functional nucleic acid based on cadmium and its application - Google Patents
A kind of visible sensor of functional nucleic acid based on cadmium and its application Download PDFInfo
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- CN107966436A CN107966436A CN201711022150.2A CN201711022150A CN107966436A CN 107966436 A CN107966436 A CN 107966436A CN 201711022150 A CN201711022150 A CN 201711022150A CN 107966436 A CN107966436 A CN 107966436A
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Abstract
Visible sensor and its application the invention discloses a kind of functional nucleic acid based on cadmium of metal ion detection technical field.The sensor includes molecular recognition elements, signal amplification component and signal conversion element, and the molecular recognition elements include cadmium ion deoxyribozyme, and cadmium ion deoxyribozyme is made of substrate chain and enzyme chain；The signal amplification component includes isothermal duplication system and hemin, and isothermal duplication system includes amplification template；The signal conversion element includes color developing agent.The visible sensor is based on cadmium ion deoxyribozyme, isothermal index iodine and tetra- serobila liquid phase sensing technologies of G structure, possess easy quick, high sensitivity, high, the resistance to high salt of specificity and the advantages such as cost is low, the Site Detection available for cadmium ion in environment.
Description
Technical field
The invention belongs to metal ion detection technical field, and in particular to a kind of visualization of functional nucleic acid based on cadmium passes
Sensor and its application.
Background technology
Cadmium is a kind of blue and white, important, transition state heavy metal element, toxic, but due to its good pliability
And inoxidizability, it is widely used in industrial production.In the 1960s, cadmium poisoning is reported in Japan for the first time, cause
The great attention of people.Research has shown that cadmium can enter organism by respiratory tract and alimentary canal, and the cadmium into body can be right
A series of vitals such as liver, kidney, bone, brain and lung cause damage, and can also cause the damage of nerve, reproduction and the system such as immune
Wound.
Liver and kidney are the most important target organs of cadmium poisoning, and m- concentration when cadmium is presented obvious to the toxicity of liver, kidney
Dependence.High concentration or Acute cadmium toxication mainly cause hepatic injury.The hepatic injury of cadmium is mainly due to cadmium by resisting with liver
The competitive activity replaced, suppress these enzymes occurs for the metal prothetic group in oxidizing ferment, causes liver radical scavenging activity to decline,
Peroxidatic reaction of lipid and oxidative stress occur for cell.Cadmium largely consume liver cell metallothionein (Metallothionein,
MT), superoxide dismutase (Superoxide Dismutase, SOD), catalase (Catalase, CAT) and gluathione
Anti-oxidant albumen, the polypeptides such as peptide peroxidase (Glutathione peroxidase, GSH-Px), cause cell oxygen radical
Scavenging activity declines, and cytoactive oxygen class (Reactive oxygen species, ROS) content increases severely, cell experience oxidation damage
The organelle structures such as wound, mitochondria and function wreck.Low concentration or chronic cadium poisoning mainly cause injury of kidney.Into body
A cadmium part exist in the form of Cd-MT reference states, Cd-MT be difficult by most of absorbed organs, but can be by proximal convoluted tubule
Chrotoplast absorbs.The cadmium of kidney is reached by glomerular filtration with blood, and rear almost all is by proximal tubular epithelial cells reabsorption.
When kidney cadmium content exceeds the cadmium sequestering power of cell, glomerular function can be seriously damaged, and diuresis, high phosphate occurs in body
The a series of symptoms such as urine, acidaminuria, glycosuria and β2-microglobulin low-molecular weight proteinuria.In addition, cadmium has strongly cell
Toxic action, the contact of of short duration cadmium can cause Apoptosis, necrosis；Long-term cadmium exposure then can inducing cell canceration, cause to swell
Knurl.
China is the first big cadmium reserves and exploitation state, while be also the first great Ge countries of consumption in the world.In recent years, with
Further utilization of the people to cadmium, Environmental Cadmium Pollution is increasingly severe, and the report on humans and animals cadmium poisoning is also continuous
Occur.Cadmium poisoning has been changed to the another big hidden danger of serious threat human health, therefore, detection and appraisal ten for cadmium
Divide important.
At present, the detection method of heavy metal cadmium mainly has flame atomic absorption method, graphite oven atomic absorption, inductive coupling
Plasma Mass Spectrometry, inductively coupled plasma mass spectrometry and anodic stripping voltammetry etc..From the point of view of specific, NITRATE BY FLAME ATOMIC is inhaled
Receipts method is easy to operate, analyze speed is fast, measure high concentration element when disturb small, signal stabilization.Graphite oven atomic absorption is sensitive,
Accurately, selectivity is good, but online test method Matrix effects are serious, is not suitable for multi-element analysid.Inductively coupled plasma
Mass spectrography high sensitivity, selectivity is good, can analyze multiple element, but expensive degree, vulnerable to pollution at the same time.Inductive coupling etc. from
Daughter mass spectrography is easy, quick, sensing instrument is simple, cheap, easily popularization, but disturbing factor is selectively poor.Anode
Stripping voltammetry high sensitivity, resolution instrument are cheap, can measure several elements at the same time, but these methods depend on large-scale instrument
Device and special messenger's operation, are restricted in being detected at the scene with field.Using the organic molecules such as ethers, more amines, cyclophane hydro carbons as
The chemical sensitisation method of representative also has certain development, but presently, there are sensitivity it is low, it is repeatable it is poor, detection need to be organic molten
The deficiencies of being carried out in agent part, the dependable with function of detection be not high.Therefore it is free of contamination, easy to be fast there is an urgent need to develop
Speed, the method for high sensitivity and high specific meet the detection needs of micro cadmium metal.
The content of the invention
The present invention is in order to solve the technical problems such as prior art medium sensitivity is low, complicated and cost is high, particular technique
Scheme is as follows：
The present invention provides a kind of visible sensor of the functional nucleic acid based on cadmium, including molecular recognition elements, signal are put
Big element and signal conversion element,
The molecular recognition elements include cadmium ion deoxyribozyme；The cadmium ion deoxyribozyme is by substrate chain and enzyme chain group
Into；
The signal amplification component includes isothermal duplication system and hemin, and the isothermal duplication system includes expanding
Increase template；
The sequence (5 ' -3 ') of the deoxyribozyme substrate chain is：CTCACGAGTCACTATrA*
GGAAGATGGCGAAACGGGGCCGG；
The sequence (5 ' -3 ') of the deoxyribozyme enzyme chain is：ATCTTCCTTCGATAGTTAAAATAGTGACTCGTGAC；
It is described amplification template sequence (5 ' -3 ') be：
CCCTACCCGCCCTACCCCCCTACCCGCCCTACCCAACTGACTCCCGGCCCCGTTTCGCCATCTTCC
The signal conversion element includes color developing agent.
The isothermal duplication system includes A systems and B systems；
The A systems include：Expand template, dNTPs and deoxyribozyme cleaved products；
The B systems include：Bst archaeal dna polymerases and its buffer solution, Nt.BstNBI nickings restriction endonuclease and its buffering are molten
Liquid.
The Bst DNA polymerase reactions buffer solution：20mM Tris-HCl,10mM(NH4)2SO4,50mM KCl,2mM
MgSO4, 0.1% polysorbas20,0.1% bovine serum albumin(BSA), pH 8.8；
The Nt.BstNBI nickings inscribe enzyme reaction buffer solution：100mM NaCl,50mM Tris-HCl,10mM
MgCl2, 300 μ g/ml trehaloses, pH 7.9.
The signal conversion element further includes terminator, it is preferred that the terminator is sulfuric acid；The color developing agent is TMB
Color developing agent.
A kind of method for detecting cadmium ion, includes the following steps：
Prepare concentration of cadmium ions and the standard curve of tetra- serobila functional nucleic acids of G- colour developing optical density (OD values) relation；
The process for being prepared as described above standard curve carries out the detection of sample to be tested, obtains the tetra- chain body functions of G- of sample to be tested
Nucleic acid colour developing OD value, passes through the concentration that above-mentioned standard curve calculates cadmium ion；
Wherein concentration of cadmium ions and tetra- serobila functional nucleic acids of G- colour developing optical density relation standard curve the step of include：
(1) various concentrations cadmium-ion solution is added in the substrate chain and enzyme chain of cadmium ion deoxyribozyme, prepares cadmium ion
Deoxyribozyme cleaved products；
The sequence (5 ' -3 ') of the deoxyribozyme substrate chain is：CTCACGAGTCACTATrA*
GGAAGATGGCGAAACGGGGCCGG；
The sequence (5 ' -3 ') of the deoxyribozyme enzyme chain is：ATCTTCCTTCGATAGTTAAAATAGTGACTCGTGAC；
(2) template, dNTPs will be expanded, cadmium ion deoxyribozyme cleaved products and ultra-pure water are uniformly mixed, and prepare A systems；
By Bst archaeal dna polymerases and its buffer solution, Nt.BstNBI nickings restriction endonuclease and its buffer solution are uniformly mixed, and prepare B bodies
System；
It is described amplification template sequence (5 ' -3 ') be：
CCCTACCCGCCCTACCCCCCTACCCGCCCTACCCAACTGACTCCCGGCCCCGTTTCGCCATCTTCC；
(3) A systems are first incubated, and are then mixed rapidly with B systems, are incubated amplification, are expanded after terminating reaction
Increase production thing；
(4) amplified production, enzyme activity buffer solution and hemin dilute solution are mixed and reacted, form tetra- serobilas of G-
Structure；
(5) nitrite ion is added into step (4) mixed liquor, mixes and react, add H after reaction2SO4, measure OD
Value.
The step of step (1) is：The substrate chain of cadmium ion deoxyribozyme and enzyme chain are diluted with buffer solution, 95 DEG C of heating
15min, is then slowly dropped to 25 DEG C；Cadmium-ion solution to be measured is added, 25 DEG C of incubation 6min, add terminate liquid, obtain cadmium ion
Deoxyribozyme cleaved products.
The step of step (3) is：A systems are incubated 5min in 95 DEG C, are then mixed rapidly with B systems, 55 DEG C of incubations
20min is expanded, 95 DEG C keep 10min to terminate reaction.
Reaction temperature is 37 DEG C in step (4), reaction time 30min；Reaction temperature is 37 DEG C in step (5), reaction
Time is 10min.
The present invention also provides a kind of kit for detecting cadmium ion at the same time, including cadmium ion deoxyribozyme system, isothermal expand
Increasing system and display system；
The cadmium ion deoxyribozyme system includes substrate chain, enzyme chain, buffer solution, cadmium ion standard solution and terminate liquid；
The buffer solution is final concentration 25mM HEPES buffer, pH 7.6；The terminate liquid is 0.2M EDTA, 2M NaCl, 0.5M
Tris；
The isothermal duplication system includes amplification template, dNTPs, ultra-pure water, Bst archaeal dna polymerases, polymeric enzyme reaction and delays
Rush solution, Nt.BstNBI nickings restriction endonuclease and Nt.BstNBI nicking inscribe enzyme reaction buffer solutions；
The display system includes：Hemin, enzyme activity buffer solution, TMB color developing agents and 2MH2SO4；The enzyme activity is delayed
Fliud flushing is 100mM Tris, 120mM NaCl, 10mM MgCl2, 100mM KCl, pH8.4；
The sequence (5 ' -3 ') of the deoxyribozyme substrate chain is：CTCACGAGTCACTATrA*
GGAAGATGGCGAAACGGGGCCGG；
The sequence (5 ' -3 ') of the deoxyribozyme enzyme chain is：ATCTTCCTTCGATAGTTAAAATAGTGACTCGTGAC；
It is described amplification template sequence (5 ' -3 ') be：
CCCTACCCGCCCTACCCCCCTACCCGCCCTACCCAACTGACTCCCGGCCCCGTTTCGCCATCTTCC。
Using the method for the kit detection cadmium ion, include the following steps：
(1) concentration of cadmium ions and the standard curve of tetra- serobila functional nucleic acids of G- colour developing optical density relation are prepared
4 μ L substrates chains of cadmium ion deoxyribozyme and 4 μ L enzymes chains are diluted to 35 μ L, 95 DEG C of heating 15min with buffer solution,
Then 25 DEG C are slowly dropped to, adds 5 μ L various concentrations cadmium-ion solutions, 25 DEG C of incubation 6min, add 5 μ L terminate liquids, prepare cadmium
Ion deoxyribozyme cleaved products；
By 6 μ L, 1 μM of amplification templates, the dNTPs of 3 μ L 2.5mM, the cadmium ion deoxyribozyme cleaved products of 61 μM of μ L
It is uniformly mixed with the ultra-pure water of 9.2 μ L, prepares A systems；By the Bst archaeal dna polymerases of 0.1 μ L 8U/ μ L, 3 μ L 10 × polymerization
Enzyme reaction buffer solution, the Nt.BstNBI nickings restriction endonuclease of 1.2 μ L 10U/ μ L and 1.5 μ L 10 × Nt.BstNBI nickings in
The reaction buffer solution of enzyme cutting is uniformly mixed, and prepares B systems；
A systems are incubated 5min in 95 DEG C, are then mixed rapidly with B systems, and 55 DEG C are incubated amplification 20min, 95 DEG C of guarantors
10min is held to terminate reaction, obtains amplified production；
By 10 μ L amplified productions, 80 μ L enzyme activity buffer solutions and 10 μ L hemins dilute solutions mix, and in 37 DEG C
30min is reacted, forms tetra- stranded structures of G-；
The hemin dilute solution is mixed according to 2 μ L hemins stostes with 1mL enzyme activity buffer solutions
Arrive；
50 μ L TMB nitrite ions are added, mixes and reacts 10min in 37 DEG C, add 50 μ L2MH after reaction2SO4, enzyme
Mark instrument measure OD450, obtain OD450The standard curve that value changes with concentration of cadmium ions；
(2) process for being prepared as described above standard curve carries out the detection of sample to be tested, and cadmium is calculated by above-mentioned standard curve
The concentration of ion.
Present invention also offers a kind of cadmium ion deoxyribozyme, the cadmium ion deoxyribozyme is by substrate chain and enzyme chain group
Into；
The sequence (5 ' -3 ') of the deoxyribozyme substrate chain is：CTCACGAGTCACTATrA*
GGAAGATGGCGAAACGGGGCCGG；
The sequence (5 ' -3 ') of the deoxyribozyme enzyme chain is：ATCTTCCTTCGATAGTTAAAATAGTGACTCGTGAC.
Beneficial effects of the present invention are：
1st, the present invention provides a kind of visible sensor and cadmium ion detection method of the functional nucleic acid based on cadmium, its base
It is made of in cadmium ion deoxyribozyme two oligonucleotide chains of substrate chain and enzyme chain, forms specific secondary structure；Determination of Trace Amount Cadmium from
Son can specific recognition cadmium ion deoxyribozyme, with reference to the enzyme chain of deoxyribozyme, and activate deoxyribozyme, cut deoxyribozyme
Substrate chain, produce cleaved products；Have and only in the presence of cleaved products, inspire isothermal index iodine (EXPAR), produce
The level-one amplification and conversion of signal, and the oligonucleotide sequence for being largely rich in guanine is generated, under hemin induction
Active tetra- stranded structures of G- are formed, catalyzing hydrogen peroxide shows green with tetramethyl benzidine, and sulfuric acid is shown after terminating reaction
Yellow, so as to produce two level amplification and conversion, changes into visual signal, can qualitatively be judged.
2nd, by the conversion of signal twice, the detection of cadmium ion can be carried out by hand-held spectrum detection instrument and determine
Amount, available for the Site Detection of cadmium ion in environment, it is excellent to possess that easy quick, high sensitivity, specificity are high and cost is low etc.
Point.
3rd, inventive sensor can resist the interference of high salt concentration, realize the detection of concentration of cadmium ions in hypersaline environment,
And keep higher specificity and sensitivity.
Brief description of the drawings
Fig. 1 be cadmium ion deoxyribozyme preparation and cleaved products verification, wherein, Lane1-Marker；Lane2- is cloudy
Property control I：Deoxyribozyme enzyme chain；Lane 3- negative control II：Deoxyribozyme substrate chain and deoxyribozyme enzyme chain, no cadmium ion；
Lane4,5,6- positives：Added respectively in the system of deoxyribozyme substrate chain and deoxyribozyme enzyme chain 15uM, 30uM,
The caddy of 45uM.
Fig. 2 is the variation diagram of amplified production in isothermal index iodine, wherein, Lane1-Marker；Lane2- is expanded
Template；Lane 3- positives；Lane4- positive controls：Amplified production.
Fig. 3 is OD450It is worth the standard curve with concentration of cadmium ions.
Embodiment
Following embodiments facilitate a better understanding of the present invention.Experiment material can pass through business unless otherwise specified in embodiment
Industry approach obtains.
The present invention is passed based on cadmium ion deoxyribozyme, isothermal index iodine (EXPAR) and tetra- serobila liquid phases of G- sensing
Sense technology, builds a kind of visible sensor.Cadmium ion deoxyribozyme is made of two oligonucleotide chains of substrate chain and enzyme chain, shape
Into specific secondary structure；Trace cadmium ion can specific recognition cadmium ion deoxyribozyme, with reference to the enzyme chain of deoxyribozyme, and
Deoxyribozyme is activated, cuts the substrate chain of deoxyribozyme；Have and only in the presence of cleaved products, inspire EXPAR amplified signals, and
Generation is largely rich in the oligonucleotide sequence of guanine；The sequence forms tetra- stranded structures of G- under hemin induction,
Class horseradish peroxidase (HRP) activity is played, catalyzing hydrogen peroxide shows green with tetramethyl benzidine, passes through hand-held spectrum
Detector is detected with quantifying.
Embodiment 1：The structure of the visible sensor of functional nucleic acid based on cadmium
1st, experiment material
Potassium chloride, sodium chloride, magnesium chloride, potassium hydrogen phosphate, disodium ethylene diamine tetraacetate, sulfuric acid, tetramethyl benzidine, chlorine are high
Iron ferroheme, caddy, urea, Nt.BstNBI nicking restriction endonucleases, Bst archaeal dna polymerases, 4- hydroxyethyl piperazineethanesulfonic acids
(HEPES), sodium hydroxide, disodium hydrogen phosphate.
2nd, sequence design
Design and synthesize cadmium ion deoxyribozyme substrate chain, deoxyribozyme enzyme chain and amplification template.Expand in template
GACTC is Nt.BstNBI nicking endonuclease recognition sequences, at four base-pairs is synthesis chain cutting (between C and A) before sequence
Site；Deoxyribozyme substrate chain end CGGCCGGG sequences are to increase and expand the combination Tm values of template；Cadmium ion is cut
Site is after the rA of deoxyribozyme substrate chain.
3rd, construction method
The construction method of the visible sensor of functional nucleic acid based on cadmium, includes the following steps：
(1) 4 μ L deoxyribozyme substrates chains (10 μM of mother liquors) and 4 μ L deoxyribozyme enzymes chains (10 μM of mother liquors) are used into buffer solution
(final concentration of 25mM HEPES buffer, pH 7.6) is diluted to 35 μ L, and 95 DEG C of heating 15min, are then slowly dropped to 25 DEG C,
About time-consuming 45min.5 μ L cadmium-ion solutions to be measured are added, form 40 μ L systems, 25 DEG C of incubation 6min, add 5 μ L terminate liquids
(0.2M EDTA, 2M NaCl, 0.5M Tris), obtains cadmium ion deoxyribozyme cleaved products.With 20% modacrylic acyl
Amine gel electrophoresis is verified, it was demonstrated that the preparation of cadmium ion deoxyribozyme is with cutting successfully, as a result such as Fig. 1.
The sequence (5 ' -3 ') of cadmium ion deoxyribozyme cleaved products is：GGAAGATGGCGAAACGGGGCCGG.
(2) amplification reaction system is prepared
Reaction system is 30 μ L, is made of part A and part B.
A systems form (24.2 μ L)
B systems form (5.8 μ L)
The "×" of the present invention is such as not particularly limited, then is measured again for volume.
" final concentration " of the present invention is not particularly limited, then is the concentration in total reaction system after material mixing.Such as 1 μM
Expand 6 μ L of template mother liquor, concentration of the final concentration of amplification template 0.2 μM, referred in isothermal duplication system.
(3) then A systems are mixed rapidly after 95 DEG C are incubated 5min with B systems, and 55 DEG C are incubated amplification 20min；95
DEG C keep 10min, with terminate reaction, obtain amplified production.Be put into -20 DEG C it is spare.Utilize 20% polyacrylamide gel electricity
Swimming verification amplified production, as a result such as Fig. 2.
The sequence (5 ' -3 ') of amplified production is GGGTAGGGCGGGTAGGGGGGTAGGGCGGGTAGGG
(4) 10 μ L amplified productions, 80 μ L enzyme activity buffer solutions and 10 μ L hemins dilute solutions are mixed, 37 DEG C anti-
30min is answered, amplified production combination hemin is formed tetra- stranded structures of G-；
Enzyme activity buffer solution：100mM Tris、120mM NaCl、10mM MgCl2, 100mM KCl, pH8.4.
Hemin dilute solution：2 μ L hemins stostes are mixed with 1mL enzyme activity buffer solutions.
(5) 50 μ L TMB nitrite ions are added into step (4) mixed liquor, are mixed, 37 DEG C of reaction 10min, add 50 μ L 2M
H2SO4, mixes, microplate reader measure OD450。
Embodiment 2：The detection of cadmium ion
Cadmium-ion solution to be measured is cadmium chloride solution (NaCl is dissolving environment) in the present embodiment, and the detection of cadmium ion is specific
Step is as follows：
(1) OD is prepared450The standard curve changed with concentration of cadmium ions
Using in embodiment 13 method, cadmium-ion solution to be measured is that (1MNaCl is dissolving to different concentrations of cadmium chloride solution
Environment), chlorination cadmium concentration takes 15uM, 30uM, 45uM, 60uM, 75uM, 90uM and 115uM, prepares OD450Become with concentration of cadmium ions
The standard curve (Fig. 3) of change, standard curve y=0.009x-0.017, R2=0.996.
(2) method for using in embodiment 13, microplate reader measure the OD of cadmium-ion solution to be measured450, bringing standard curve into is
Y=0.009x-0.017, obtains concentration of cadmium ions.Correlated results such as table 1.
Table 1
Embodiment 3：A kind of kit for detecting cadmium ion
A kind of kit for detecting cadmium ion, including cadmium ion deoxyribozyme system, isothermal duplication system and display system；
Cadmium ion deoxyribozyme system includes substrate chain, enzyme chain, buffer solution, cadmium ion standard solution and terminate liquid；
It is molten that isothermal duplication system includes amplification template, dNTPs, ultra-pure water, Bst archaeal dna polymerases, polymeric enzyme reaction buffering
Liquid, Nt.BstNBI nickings restriction endonuclease and Nt.BstNBI nicking inscribe enzyme reaction buffer solutions；
Display system includes：Hemin, enzyme activity buffer solution, TMB color developing agents and 2MH2SO4。
The sequence (5 ' -3 ') of deoxyribozyme substrate chain is：CTCACGAGTCACTATrA*GGAAGATGGCGAAACGGGGCC
GG；
The sequence (5 ' -3 ') of deoxyribozyme enzyme chain is：ATCTTCCTTCGATAGTTAAAATAGTGACTCGTGAC；
Amplification template sequence (5 ' -3 ') be：CCCTACCCGCCCTACCCCCCTACCCGCCCTACCCAACTGACTCCC
GGCCCCGTTTCGCCATCTTCC。
Buffer solution is final concentration 25mM HEPES buffer, pH 7.6；
Terminate liquid is 0.2M EDTA, 2M NaCl, 0.5M Tris；
Enzyme activity buffer solution is 100mM Tris, 120mM NaCl, 10mM MgCl2, 100mM KCl, pH8.4.
Bst DNA polymerase reaction buffer solutions：20mM Tris-HCl,10mM(NH4)2SO4,50mMKCl,2mM MgSO4,
0.1% polysorbas20,0.1% bovine serum albumin(BSA), pH 8.8.
Nt.BstNBI nicking inscribe enzyme reaction buffer solutions：100mM NaCl,50mM Tris-HCl,10mMMgCl2,300μ
G/ml trehaloses, pH 7.9.
Claims (10)
1. a kind of visible sensor of the functional nucleic acid based on cadmium, including molecular recognition elements, signal amplification component and signal
Conversion element, it is characterised in that
The molecular recognition elements include cadmium ion deoxyribozyme；The cadmium ion deoxyribozyme is made of substrate chain and enzyme chain；
The signal amplification component includes isothermal duplication system and hemin, and the isothermal duplication system includes amplification mould
Plate；
The sequence (5 ' -3 ') of the deoxyribozyme substrate chain is：CTCACGAGTCACTATrA*
GGAAGATGGCGAAACGGGGCCGG；
The sequence (5 ' -3 ') of the deoxyribozyme enzyme chain is：ATCTTCCTTCGATAGTTAAAATAGTGACTCGTGAC；
It is described amplification template sequence (5 ' -3 ') be：
CCCTACCCGCCCTACCCCCCTACCCGCCCTACCCAACTGACTCCCGGCCCCGTTTCGCCATCTTCC；
The signal conversion element includes color developing agent.
2. sensor according to claim 1, it is characterised in that the isothermal duplication system includes A systems and B systems；
The A systems include：Expand template, dNTPs and cadmium ion deoxyribozyme cleaved products；
The B systems include：Bst archaeal dna polymerases and its buffer solution, Nt.BstNBI nickings restriction endonuclease and its buffer solution.
3. sensor according to claim 1 or 2, it is characterised in that the signal conversion element further includes terminator, excellent
Choosing, the terminator is sulfuric acid；The color developing agent is TMB color developing agents.
A kind of 4. method for detecting cadmium ion, it is characterised in that include the following steps：
Prepare concentration of cadmium ions and the standard curve of tetra- serobila functional nucleic acids of G- colour developing optical density relation；
The process for being prepared as described above standard curve carries out the detection of sample to be tested, obtains the tetra- serobila functional nucleic acids of G- of sample to be tested
Develop the color OD value, and the concentration of cadmium ion is calculated by above-mentioned standard curve；
Wherein, concentration of cadmium ions and tetra- serobila functional nucleic acids of G- colour developing optical density relation standard curve the step of include：
(1) various concentrations cadmium-ion solution is added in the substrate chain and enzyme chain of cadmium ion deoxyribozyme, prepares cadmium ion deoxidation
Ribozyme cleaved products；
The sequence (5 ' -3 ') of the deoxyribozyme substrate chain is：CTCACGAGTCACTATrA*
GGAAGATGGCGAAACGGGGCCGG；
The sequence (5 ' -3 ') of the deoxyribozyme enzyme chain is：ATCTTCCTTCGATAGTTAAAATAGTGACTCGTGAC；
(2) template, dNTPs will be expanded, cadmium ion deoxyribozyme cleaved products and ultra-pure water are uniformly mixed, and prepare A systems；Will
Bst archaeal dna polymerases and its buffer solution, Nt.BstNBI nickings restriction endonuclease and its buffer solution are uniformly mixed, and prepare B systems；
It is described amplification template sequence (5 ' -3 ') be：
CCCTACCCGCCCTACCCCCCTACCCGCCCTACCCAACTGACTCCCGGCCCCGTTTCGCCATCTTCC；
(3) A systems are first incubated, and are then mixed rapidly with B systems, are incubated amplification, and amplification production is obtained after terminating reaction
Thing；
(4) amplified production, enzyme activity buffer solution and hemin dilute solution are mixed and reacted, form tetra- serobila knots of G-
Structure；
(5) nitrite ion is added into step (4) mixed liquor, mixes and react, add H after reaction2SO4, measure OD450Value,
Obtain OD450The standard curve that value changes with concentration of cadmium ions.
5. according to the method described in claim 4, it is characterized in that, the operation of step (1) is：By the bottom of cadmium ion deoxyribozyme
Thing chain and enzyme chain are diluted with buffer solution, and 95 DEG C of heating 15min, are then slowly dropped to 25 DEG C；Add cadmium-ion solution to be measured, 25 DEG C
6min is incubated, terminate liquid is added, obtains cadmium ion deoxyribozyme cleaved products.
6. according to the method described in claim 4, it is characterized in that, the operation of step (3) is：A systems are incubated 5min in 95 DEG C,
Then mixed rapidly with B systems, 55 DEG C are incubated amplification 20min, and 95 DEG C keep 10min to terminate reaction.
7. according to the method described in claim 4, it is characterized in that, reaction temperature is 37 DEG C in step (4), the reaction time is
30min；Reaction temperature is 37 DEG C in step (5), reaction time 10min.
8. it is a kind of detect cadmium ion kit, it is characterised in that including cadmium ion deoxyribozyme system, isothermal duplication system and
Display system；
The cadmium ion deoxyribozyme system includes substrate chain, enzyme chain, buffer solution, cadmium ion standard solution and terminate liquid；
It is molten that the isothermal duplication system includes amplification template, dNTPs, ultra-pure water, Bst archaeal dna polymerases, polymeric enzyme reaction buffering
Liquid, Nt.BstNBI nickings restriction endonuclease and Nt.BstNBI nicking inscribe enzyme reaction buffer solutions；
The display system includes：Hemin, enzyme activity buffer solution, TMB color developing agents and 2MH2SO4；
The sequence (5 ' -3 ') of the deoxyribozyme substrate chain is：CTCACGAGTCACTATrA*
GGAAGATGGCGAAACGGGGCCGG；
The sequence (5 ' -3 ') of the deoxyribozyme enzyme chain is：ATCTTCCTTCGATAGTTAAAATAGTGACTCGTGAC；
It is described amplification template sequence (5 ' -3 ') be：
CCCTACCCGCCCTACCCCCCTACCCGCCCTACCCAACTGACTCCCGGCCCCGTTTCGCCATCTTCC。
9. kit according to claim 8, it is characterised in that the buffer solution is final concentration 25mM HEPES
Buffer, pH 7.6；The terminate liquid is 0.2M EDTA, 2M NaCl, 0.5M Tris；The enzyme activity buffer solution is 100mM
Tris、120mM NaCl、10mM MgCl2, 100mM KCl, pH8.4.
10. a kind of cadmium ion deoxyribozyme, it is characterised in that the cadmium ion deoxyribozyme is made of substrate chain and enzyme chain；
The sequence (5 ' -3 ') of the deoxyribozyme substrate chain is：CTCACGAGTCACTATrA*
GGAAGATGGCGAAACGGGGCCGG；
The sequence (5 ' -3 ') of the deoxyribozyme enzyme chain is：ATCTTCCTTCGATAGTTAAAATAGTGACTCGTGAC.
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Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108841937A (en) * | 2018-06-20 | 2018-11-20 | 中国农业大学 | It is general to separate ultrafast amplification magnesium, zinc cutting-type functional nucleic acid visible detection method |
| CN108841936A (en) * | 2018-06-20 | 2018-11-20 | 中国农业大学 | A kind of general ultrafast amplification visible sensor of partition of calcium ion cutting-type |
| CN108929900A (en) * | 2018-06-20 | 2018-12-04 | 中国农业大学 | A kind of general ultrafast amplification visible sensor of partition of cadmium ion cutting-type |
| CN108949933A (en) * | 2018-06-20 | 2018-12-07 | 中国农业大学 | A kind of general ultrafast amplification colorimetric sensor of partition of silver ion mispairing type |
| CN108949935A (en) * | 2018-06-20 | 2018-12-07 | 中国农业大学 | It is general to separate ultrafast amplification chromium, cadmium cutting-type functional nucleic acid visible detection method |
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| CN112011597A (en) * | 2020-07-24 | 2020-12-01 | 南京师范大学 | Cadmium ion sensing method combining induced allosteric probe with rolling circle amplification |
| CN112924406A (en) * | 2021-02-02 | 2021-06-08 | 湘潭大学 | Mimic enzyme-assisted mercury ion detection method and kit |
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Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1272062A (en) * | 1997-07-17 | 2000-11-01 | 北美疫苗公司 | Immunogenic conjugates comprising group meningococcal porin and an i (H. influenzae) polysaccharide |
| CN1850981A (en) * | 2006-03-10 | 2006-10-25 | 杭州优思达生物技术有限公司 | Method for amplifying target nucleic acid sequence by nickase, and kit for amplifying target nucleic acid sequence and its use |
| CN103163127A (en) * | 2013-03-06 | 2013-06-19 | 上海交通大学 | Method for detecting trivalent arsenic by protoheme horseradish peroxidase catalytic colorimetry |
| CN103305612A (en) * | 2013-06-04 | 2013-09-18 | 西安交通大学 | Lead ion detection kit based on constant-temperature cascade nucleic acid amplification and detection method of lead ion detection kit |
| WO2015164957A1 (en) * | 2014-04-28 | 2015-11-05 | Juewen Liu | Phosphorothioate dnazyme complexes and use thereof |
| CN107012208A (en) * | 2017-03-08 | 2017-08-04 | 广东省生态环境技术研究所 | A kind of label-free lead ion visible detection method and detection kit |
| CN107217100A (en) * | 2017-06-29 | 2017-09-29 | 中国农业大学 | Nucleic acid screening biology sensor based on DNA enzymatic chimeric primers |
| CN108318479A (en) * | 2017-12-23 | 2018-07-24 | 张睿 | A kind of lead ion detection method of high sensitivity |
-
2017
- 2017-10-27 CN CN201711022150.2A patent/CN107966436B/en active Active
Patent Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1272062A (en) * | 1997-07-17 | 2000-11-01 | 北美疫苗公司 | Immunogenic conjugates comprising group meningococcal porin and an i (H. influenzae) polysaccharide |
| CN1850981A (en) * | 2006-03-10 | 2006-10-25 | 杭州优思达生物技术有限公司 | Method for amplifying target nucleic acid sequence by nickase, and kit for amplifying target nucleic acid sequence and its use |
| CN103163127A (en) * | 2013-03-06 | 2013-06-19 | 上海交通大学 | Method for detecting trivalent arsenic by protoheme horseradish peroxidase catalytic colorimetry |
| CN103305612A (en) * | 2013-06-04 | 2013-09-18 | 西安交通大学 | Lead ion detection kit based on constant-temperature cascade nucleic acid amplification and detection method of lead ion detection kit |
| WO2015164957A1 (en) * | 2014-04-28 | 2015-11-05 | Juewen Liu | Phosphorothioate dnazyme complexes and use thereof |
| US20170241971A1 (en) * | 2014-04-28 | 2017-08-24 | Juewen Liu | Phosphorothioate dnazyme complexes and use thereof |
| CN107012208A (en) * | 2017-03-08 | 2017-08-04 | 广东省生态环境技术研究所 | A kind of label-free lead ion visible detection method and detection kit |
| CN107217100A (en) * | 2017-06-29 | 2017-09-29 | 中国农业大学 | Nucleic acid screening biology sensor based on DNA enzymatic chimeric primers |
| CN108318479A (en) * | 2017-12-23 | 2018-07-24 | 张睿 | A kind of lead ion detection method of high sensitivity |
Non-Patent Citations (4)
| Title |
|---|
| LIBING ZHANG等: ""Bifunctional Colorimetric Oligonucleotide Probe Based on a G-Quadruplex DNAzyme Molecular Beacon"", 《ANALYTICAL》 * |
| PO-JUNG JIMMY HUANG 等: ""A new heavy lanthanide-dependent DNAzyme displaying strong metal cooperativity and unrescuable phosphorothioate effect"", 《NUCLEIC ACIDS RESEARCH》 * |
| PO-JUNG JIMMY HUANG等: ""Sensing Parts-per-Trillion Cd2+,Hg2+, and Pb2+ Collectively and Individually Using Phosphorothioate DNAzymes"", 《ANALYTICAL CHEMISTRY》 * |
| TAO LI 等: ""Lead(II)-Induced Allosteric G-Quadruplex DNAzyme as a Colorimetric and Chemiluminescence Sensor for Highly Sensitive and Selective Pb2 Detection"", 《ANAL.CHEM.》 * |
Cited By (16)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108949935B (en) * | 2018-06-20 | 2020-07-28 | 中国农业大学 | Universal partition ultrafast amplification chromium and cadmium cutting type functional nucleic acid visual detection method |
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| CN108841937B (en) * | 2018-06-20 | 2020-07-28 | 中国农业大学 | General partition ultrafast amplification magnesium and zinc cutting type functional nucleic acid visual detection method |
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| CN108841936B (en) * | 2018-06-20 | 2020-10-02 | 中国农业大学 | Calcium ion cutting type universal partition ultrafast amplification visual sensor |
| CN112011597A (en) * | 2020-07-24 | 2020-12-01 | 南京师范大学 | Cadmium ion sensing method combining induced allosteric probe with rolling circle amplification |
| CN112011597B (en) * | 2020-07-24 | 2023-03-21 | 南京师范大学 | Cadmium ion sensing method combining induced allosteric probe with rolling circle amplification |
| CN112924406A (en) * | 2021-02-02 | 2021-06-08 | 湘潭大学 | Mimic enzyme-assisted mercury ion detection method and kit |
| CN112924406B (en) * | 2021-02-02 | 2022-08-12 | 湘潭大学 | Mimic enzyme-assisted mercury ion detection method and kit |
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